Activated neutrophils (PMNs) play a critical role in sepsis, ischemia-reperfusion injury, and immune complex-mediated diseases. The broad long-term objective of our laboratory is to elucidate the role of Akt in regulating PMN functions. This proposal will test the hypothesis that Hsp27 modulates neutrophil apoptosis by functioning as a scaffolding protein for Akt activation and induction of neutrophil survival pathways. The specific aims of this proposal are: Specific Aim 1-To identify proteins whose association with the Akt signaling complex is regulated by Hsp27.Specific Aim 2-To determine the effect of protein changes, following disruption of Akt-Hsp27complex, on the regulation of Akt activation and neutrophil apoptosis. Specific Aim 3 -To identify signaling pathways that underlie Akt-Hsp27 disruption-induced neutrophil apoptosis. Specific Aim 4- To identify Akt auto-phosphorylation sites and determine their effect on assembly of Akt-Hsp27 signal complex, Akt activation, and neutrophil apoptosis. In specific aim 1 we will disrupt Akt/Hsp27 interaction by 3 separate approaches. A) By transducing TAT-scrambled or TAT- Akt117-128 peptide, which encompasses Hsp27 binding site on Akt. B) We will introduce control or anti-Hsp27 antibody into PMNs. C) Transfecting HK-11 cells with scrambled or Hsp27 siRNA. Protein lysates will be subjected to Akt and Hsp27 immunoprecipitation, 2D SDS-PAGE, MALDI-MS, and LC-ESI-MS/MS analysis to identify proteins changes within Akt-Hsp27 complex in the presence or absence of Akt-Hsp27 interaction. Specific aim 2 experiments will determine if proteins (identified in specific aim 1) that associate/ dissociate from Akt-Hsp27 complex, affect Akt activity and neutrophil apoptosis. In specific aim 3 we will identify signaling cascades that induce neutrophil apoptosis in the absence of Akt-Hsp27 interaction. In specific aim 4 site-directed mutagenesis and mass spectroscopic techniques will be used to identify in vitro Akt auto-phosphorylation sites. Phospho-specific antibodies to Akt auto-phosphorylation sites will be generated commercially to identify in vivo phosphorylation sites in neutrophils and to determine its effect on assembly of Akt signal module, Akt activation, and neutrophil apoptosis. These studies will lead us to new targets for disrupting neutrophil-mediated tissue damage in inflammatory diseases. [unreadable] [unreadable] [unreadable]